The present invention concerns peptides as inhibitors of the binding of urokinase to the urokinase receptor. These peptides which are preferably cyclic are suitable as pharmaceutical agents for diseases which are mediated by urokinase and its receptor.
The serine protease uPA (urokinase-type plasminogen activator) is responsible for various physiological and pathological processes such as the proteolytic degradation of extracellular matrix material which is necessary for the invasiveness and migration of cells and for tissue remodelling. uPA binds with high affinity (KD=10xe2x88x9210xe2x88x9210xe2x88x929 M) to the membrane-based uPA receptor (uPAR) on the cell surface.
The binding of uPA to its receptor is involved in many invasive biological processes such as the metastatic spread of malignant tumours, trophoplast implantation, inflammation and angiogenesis. Hence antagonists of uPA are able to inhibit the invasiveness, metastatic spread and angiogenesis of tumours. uPA antagonists can be used as agents for the treatment of invasive and metastasising cancer diseases in which uPA and uPAR occur at the invasive foci of tumours (Dano et al., The receptor for urokinase plasminogen activator: Stromal cell involvement in extracellular proteolysis during cancer invasion, in: Proteolysis and Protein Turnover, Barrett, A. J. and Bond, J., Editor, Portland Press, London, 1994, 239) e.g. in cancers of the breast, lung, intestine and ovaries. In addition uPA antagonists can also be used for other purposes in which it is necessary to inhibit the proteolytic activation of plasminogen, for example to treat diseases such as arthritis, inflammation, osteoporosis, retinopathies and for contraception.
The uPA receptor is described in WO 90/12091 and in the publications by Ploug et al., J. Biol. Chem. 268 (1993), 17539 and Ronne et al., J. Immunol. Methods 167 (1994), 91.
uPA is synthesized as a single chain molecule (pro-uPA) and is converted enzymatically into an active two-chain uPA. The uPA molecule is composed of three structurally independent domains, the N-terminal growth factor-like domain (GFD, uPA 1-46), a kringle structure domain (uPA 45-135) and the serine protease domain (uPA 159-411). GFD and the kringle domain together form the so-called aminoterminal fragment of uPA (ATF, uPA 1-135) which is produced by further proteolytic cleavage of two-chain uPA. ATF binds to the uPA receptor with a similar affinity to uPA.
The receptor-binding region of uPA spans the region of the amino acids 12 to 32 since a peptide which contains the amino acid residues 12 to 32 of uPA (in which case cysteine is replaced by alanine in position 19) competes with ATF for binding to the uPA receptor (Appella et al., J. Biol. Chem. 262 (1987), 4437-4440). In this publication it was also shown that this peptide also has an affinity for the uPA receptor after cyclization by bridging the two cysteine residues at positions 12 and 32. In an alternative approach Goodson et al., (Proc. Natl. Acad. USA 91 (1994), 7129-7133) identified antagonistic uPA peptides for the uPAR by screening a bacteriophage peptide library. These peptides had no apparent sequence homology to the natural uPAR-binding sequence of uPA.
Further investigations of the uPAR-binding region of uPA are described in recent publications (Rettenberger et al., Biol. Chem. Hoppe-Seyler 376 (1995), 587-594); Magdolen et al., Eur. J. Biochem. 237 (1996), 743-751; Goretzki et al., Fibrinolysis and Proteolysis 11 (1997), 11-19). The residues Cysl9, Lys23, Tyr24, Phe25, Ile28, Trp30 and Cys31 were identified as important determinants for a uPA/uPAR interaction. In these investigations a uPA peptide having the amino acids 16 to 32 of uPA was identified as the most effective inhibitor.
Magdolen et al., (1996) supra analysed the uPAR binding region of the uPA molecule using a peptide having the amino acids 14 to 32 of uPA and peptides derived therefrom. However, these peptides and also peptides used by other research groups (cf. e.g. Appella et al., (1987) supra) have a relatively low affinity for uPAR.
WO-A-94/22464 discloses peptides with a length of 6 to 18 amino acids which are derived from the region of the amino acids 14 to 33 of uPA. It is described that short peptides derived from uPA (uPA 21-29 and uPA 21-26) are able to influence the growth of keratinocytes. Although WO-A-94/22464 makes reference to a potential use of the claimed peptides to block the uPA/uPAR interaction, no data or information whatsoever is shown on such binding studies. Moreover, the peptides uPA 21-29 and uPA 21-26 which are said to be preferred do not contain the minimal uPAR binding region in the uPA molecule which comprises the sequence region of amino acids 19 to 31. Hence the influence of the growth of keratinocytes by these short peptides is very probably not due to a uPA/uPAR interaction.
A disadvantage of the previously known uPA peptide inhibitors is that the affinity of the binding to the uPA receptor is relatively low and inadequate for a therapeutic application. Thus there is a great need for new uPA peptide antagonists which have a higher affinity for the receptor.
The present invention provides a peptide having the general structural formula (I):
X1-[X2]n-X3-X4-K-Y-F-X5-X6-I-X7-W-[X8]mxe2x80x83xe2x80x83(I)
in which
X1, X2, X3, X4, X5, X6, X7 and X8 each denote an aminocarboxylic acid,
n and m are each independently 0 or 1,
K denotes an aminocarboxylic acid with a lysine side chain,
Y denotes an aminocarboxylic acid with a tyrosine side chain,
F denotes an aminocarboxylic acid with a phenyl-alanine side chain,
I denotes an aminocarboxylic acid with an isoleucine side chain,
W denotes an aminocarboxylic acid with a tryptophan side chain,
and the monomeric building blocks are linked by xe2x80x94CONR1xe2x80x94 or xe2x80x94NR1COxe2x80x94 bonds where R1 in each case independently denotes hydrogen, methyl or ethyl, and pharmaceutically compatible salts and derivatives thereof.
The present invention also provides a pharmaceutical composition comprising at least one peptide of formula I as an active substance.
The present invention further provides the use of a peptide of formula I as a urokinase (uPA) antagonist.
The present invention further provides the treatment of a tumor by the use of a peptide of formula I.
In quantitative investigations it was surprisingly found that the linear peptide uPA (19-31) and cyclic derivatives of this peptide have a considerably improved binding affinity for the uPA receptor.
Experimental data have shown that the peptides according to the invention can be used as uPA antagonists which bind with high affinity to the uPAR. Cyclic peptides are particularly preferred which are characterized by bridges, especially disulfide bridges which do not occur in the native uPA molecule.
Hence the present invention concerns peptides having the general structural formula (I):
X1-[X2 ]n-X3-X4-K-Y-F-X5-X6-I-X7-W-[X8]mxe2x80x83xe2x80x83(I)
in which
X1, X2, X3, X4, X5, X6, X7 and X8 each denote an amino-carboxylic acid, preferably an a-aminocarboxylic acid,
n and m are each independently 0 or 1,
K denotes an aminocarboxylic acid, preferably an xcex1-aminocarboxylic acid with a lysine side chain,
Y denotes an aminocarboxylic acid, preferably an xcex1-aminocarboxylic acid with a tyrosine side chain,
F denotes an aminocarboxylic acid, preferably an xcex1-aminocarboxylic acid with a phenylalanine side chain,
I denotes an aminocarboxylic acid, preferably an xcex1-aminocarboxylic acid with an isoleucine side chain,
W denotes an aminocarboxylic acid, preferably an xcex1-aminocarboxylic acid with a tryptophan side chain,
and the monomeric building blocks are linked by xe2x80x94CONR1xe2x80x94 or xe2x80x94NR1COxe2x80x94 bonds in where R1 in each case independently denotes hydrogen, methyl or ethyl, and pharmaceutically compatible salts and derivatives thereof.
In addition to peptides which contain a sequence having the structural formula (I), pharmaceutically compatible salts and derivatives thereof are also suitable as uPA antagonists. Suitable derivatives are in particular compounds in which the reactive groups of the side chain or/and of the N-terminus or C-terminus e.g. amino or carboxylic acid groups have been modified. Examples of such modifications are acylation e.g. an acetylation of amino groups or/and an amidation or esterification of carboxylic acid groups.
Natural amino acids or enantiomers thereof or non-natural amino acids i.e. amino acids that are not genetically coded such as xcex3-aminobutyric acid, xcex2-alanine can be used as building blocks for the peptides according to the invention.
The monomeric building blocks are linked by acid amide bonds NR1CO or CONR1 i.e. the direction of the peptide sequence can also be reversed (retropeptides). As in native polypeptides, R1 can denote hydrogen. On the other hand, R1 can also denote an alkyl residue e.g. methyl or ethyl and in particular methyl since N-alkylation of the amide bond often has a major influence on the activity (cf. e.g. Levian-Teitelbaum et al., Biopolymers 28 (1989), 51-64).
The xcex1-aminocarboxylic acids can also be used as monomeric building blocks in the form of L-enantiomers or/and D-enantiomers. The spatial structure of the peptides according to the invention can be modified by changing the chirality which can also influence the activity. Retro-inverso peptides are particularly preferred i.e. peptides which are present in a reversed sequence direction and contain D-amino acids as monomeric building blocks. In these D-inverso structures the functional side chains have a similar spatial orientation to those in the native peptide sequence, but their biological degradation is impaired due to the presence of D-amino acids and they therefore have advantages as drugs (cf. for example Wermuth et al., J. Am. Chem. Soc. 119 (1997), 1328-1335 and references cited therein).
The peptides according to the invention are preferably cyclic compounds in which in particular the monomeric building blocks X1 and X7 and X1 and X8 are bridged together. This bridge is preferably not a natural bridge i.e. a bridge which does not occur in natural uPA. It can for example utilize the side chains of the respective xcex1-aminocarboxylic acid residues in which case bridging by means of disulfide bonds e.g. between two cysteine residues (corresponding to a bridge between Cys19 and Cys31 of the natural uPA sequence) is particularly preferred. Other types of cyclization between amino acid side chains are, however, also possible e.g. amide bonds between an amino acid with an xcfx89 amino side group e.g. Lys and an amino acid with a carboxylic acid side group such as Asp or Glu. In addition the disulfide bridge can also be replaced by an alkylene bridge in order to increase the chemical stability. In addition an amino acid side chain may also be linked to the peptide backbone e.g. an omega amino side group may be linked with the C-terminal end or a carboxylic acid side group may be linked with the N-terminal end. A linkage of the N-terminus and C-terminus is also possible.
Instead of the disulfide bridge it is also possible to use so-called turn mimetics (Haubner et al., J. Am. Chem. Soc. 118 (1996), 7884-7891) or sugar amino acids (Graf von Rbdern et al., J. Am. Chem. Soc. 118 (1996), 10156-10167).
In a particularly preferred embodiment of the present invention the peptides have the general structural formula (II):
X1-X2-X3-X4-K-Y-F-X5-X6-I-X7-W-X8xe2x80x83xe2x80x83(II)
in which X1, X2, X3, X4, X5, X6, X7, X8, K, Y, F, I and W are defined as above and X1 and X8 are bridged together.
In yet a further preferred embodiment the peptides according to the invention have the general structural formula (III):
xe2x80x83X1-X3-X4-K-Y-F-X5-X6-I-X7-Wxe2x80x83xe2x80x83(III)
in which X1, X3, X4, X5, X6, X7, K, Y, F, I and W are defined as above and X1 and X7 are bridged together.
The monomeric building blocks X1 to X8 preferably have the following meanings:
X1 andxe2x80x94if presentxe2x80x94X8 are xcex1-aminocarboxylic acid building blocks with an SH side chain, in particular with a cysteine side chain.
X2xe2x80x94if presentxe2x80x94is preferably an xcex1-aminocarboxylic acid with an aliphatic and uncharged side chain e.g. valine, leucine or isoleucine, in particular valine.
X3xe2x80x94if presentxe2x80x94and X5 are xcex1-aminocarboxylic acids with an aliphatic hydrophilic side chain such as serine or threonine, in particular serine.
X4 and X6 are preferably xcex1-aminocarboxylic acids with an aliphatic hydrophilic side chain, in particular an amide side chain such as asparagine or glutamine, in particular asparagine.
In compounds having the structural formula (II) X7 is preferably a basic xcex1-aminocarboxylic acid, in particular histidine. In compounds of the structural formula (III) X7 is an xcex1-aminocarboxylic acid with an SH side group, in particular cysteine.
The present invention additionally concerns a pharmaceutical composition which contains at least one peptide as defined above as the active substance optionally together with common pharmaceutical carriers, auxiliary agents or diluents. The peptides according to the invention are used especially to produce uPA antagonists which are suitable for treating tumours.
The pharmaceutical compositions according to the invention can be present in any form, for example as tablets, as coated tablets or in the form of solutions or suspensions in aqueous or non-aqueous solvents. The peptides are preferably administered orally or parenterally in a liquid or solid form. When they are administered in a liquid form, water is preferably used as the carrier medium which optionally contains stabilizers, solubilizers or/and buffers that are usually used for injection solutions. Such additives are for example tartrate or borate buffer, ethanol, dimethyl sulfoxide, complexing agents such as EDTA, polymers such as liquid polyethylene oxide etc.
If they are administered in a solid form, then solid carrier substances can be used such as starch, lactose, mannitol, methyl cellulose, talcum, highly dispersed silicon dioxide, high molecular fatty acids such as stearic acid, gelatin, agar, calcium phosphate, magnesium stearate, animal and vegetable fats or solid high molecular polymers such as polyethylene glycols. The formulations can also contain flavourings and sweeteners if desired for oral administration.
The administered dose depends on the age, state of health and weight of the patient, on the type and severity of the disease, on the type of treatment, the frequency of the administration and the type of desired effect. The daily dose of the active compound is usually 0.1 to 50 mg/kilogramme body weight. Normally 0.5 to 40 and preferably 1.0 to 20 mg/kg/day in one or several doses are adequate to achieve the desired effects.